Phosphodiesterase type 3 (PDE3) exists as two subtypes: PDE3A and PDE3B, each with distinct cellular and subcellular locations. We are examining the subcellular localization of adipocyte and myocardial PDE3 isoforms and their contributions to regulation of cAMP signaling pathways. Approximately 30% of total membrane PDE3 activity is associated with plasma membrane/caveolae (PM);70%, with internal membrane (Golgi/ sarcoplasmic reticulum) fractions from failing human heart myocardium. Of the three PDE3A isoforms, PDE3A3 is the dominant PDE3A isoform in PM/caveolae fractions, while PDE3B and all isoforms of PDE3A (A1-3) are present in internal membrane fractions. PDE3A2 and PDE3A3 predominate in cytosolic fractions. Confocal microscopy indicates that both PDE3A and SERCA2 are detected in Z-band regions. PDE3A and SERCA2 were co-immunoprecipitated from human myocardial membranes using anti-PDE3A and anti-SERCA2 antibodies or cAMP-agarose beads;cilostamide, a specific PDE3 inhibitor, augmented the stimulatory effect of cAMP on Ca uptake into human myocardial microsomes. PDE3A and SERCA2 may be components of a multimolecular complex that regulates cAMP-induced Ca transients and uptake into cardiac sarcoplasmic reticulum. In adipocytes, PDE3B is an important regulatory effector of signaling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the B3-receptor agonist CL316243 (CL) demonstrated that insulin or CL preferentially phosphorylated /activated PDE3B associated with internal membranes (endoplasmic reticulum/golgi) or caveolae, respectively. siRNA-mediated knock-down (KD) of caveolin-1 (Cav-1) in 3T3-L1 adipocytes resulted in down-regulation of expression and activity of membrane- associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Cav-1 KD resulted also in inhibition of CL-stimulated phosphorylation of hormone sensitive lipase and perilipin A, and of lipolysis. Superose 6 gel filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contained 32P-phosphorylated PDE3B and signaling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signaling proteins (14-3-3, PP2A, cav-1, perilipin) as well as ligand-specific proteins. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by cav-1 KD. These data suggest that cav-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin. WAT, a highly regulated and dynamic secretory organ, affects body fat and energy utilization via storage and turnover/hydrolysis of triglycerides. In addition, via production of endocrine factors, WAT regulates and integrates important physiological pathways and homeostatic functions, including satiety, energy utilization, peripheral insulin sensitivity, glucose homeostasis, and systemic inflammatory responses. Obesity is a major risk factor for developing type 2 diabetes and cardiovascular disease. As obesity develops, there is a progressive increase in macrophages and inflammation-related effectors in WAT. Thus, WAT contributes not only to modulation of energy utilization and homeostasis, but also to metabolic dysregulation and inflammation that characterizes insulin resistance and obesity-related metabolic and cardiovascular complications. Aquirement of BAT characteristics by WAT, with enhanced intra-adipocyte FAO (fatty acid oxidation), represents a potential new strategy in treatment of obesity and diabetes. Although mechanisms for acute activation of PDE3B have been studied in isolated adipocytes and cultured cells, its role(s)in human and animal physiology, especially with regard to energy homeostatic mechanisms, is not well understood. To evaluate these functions, we introduced a targeted disruption in the murine PDE3B gene by homologous recombination (in SvJ129 background). In PDE3B KO mice, epididymal WAT (EWAT) assumed some phenotypic characteristics of BAT, including changes in morphology (increased vascularization and mitochondria number), and activation of cAMP/PKA and AMP-activated protein kinase (AMPK)-signaling pathways, as well as increased mitochondrial biogenesis and expression of genes important in differentiation of BAT, including PDRM16 and LRP130. In KO EWAT, there is coordinate regulation of expression of genes, transcriptional regulators, and mitochondrial proteins required for energy dissipation and fatty acid oxidation, such as PGC-1, PPAR, UCP-1 and its regulator CIDEA, and other mitochondrial proteins involved in election transport and fatty acid -oxidation. UCP-1, a marker for brown adipose tissue (BAT) usually not present in EWAT, is markedly elevated in EWAT from KO mice. Furthermore, in EWAT from PDE3B KO mice, phospho-LKB1, AMP kinase 1 subunits, and AMP kinase enzymatic activity were increased, as were phosphorylated substrates of AMPK, including phospho-ACC and HSL, and serum adiponectin in KO mice. Alterations in cAMP/PKA- and AMP kinase-signaling pathways were reflected in alterations in lipolysis and FAO, in that the antilipolytic actions of insulin were inhibited and FAO was increased in isolated adipocytes from KO mice. These findings most likely contribute to several phenotypic characteristics of PDE3B KO mice, including a smaller increase in body weight in response to high fat diets, smaller gonadal fat deposits and smaller adipocytes, increased oxygen consumption in vivo in response to 3 agonist stimulation, increased oxygen consumption in isolated brown and white adipose tissue fragments, and increased fatty acid oxidation in adipocytes from PDE3B KO mice. Taken together, these surprising results suggested an entirely new role for PDE3B regarding mitochondrial function, energy dissipation, adipocyte metabolism, and perhaps, that PDE3B may regulate a molecular switch for WAT/BAT phenotypic conversion, and thereby could play an important role in regulation of energy metabolism. Interestingly, we found that targeted disruption of PDE3B was associated with decreased expression of inflammatory effectors and macrophage markers in epididymal white adipose tissue (EWAT), including MCP-1, macrophage inflammatory protein-1 (MIP1/Ccl3), Ccr2 and Ccr5, Adam8, F4/80 (Emr1), activating transcription factor 3 (Atf3) (a stress-inducible transcriptional factor), as well as cell adhesion molecules. Moreover, chemokine (C-C motif) ligand 2 (CCL2) and its receptor CCR2, which play an important role in macrophage chemotaxis, were less highly expressed in EWAT of PDE3B-/- mice than WT mice. Accumulation of macrophages in WT EWAT, associated with high fat feeding, was reduced in KO EWAT, consistent with reduced proinflammatory molecules in KO EWAT. In addition, after lipopolysaccaride (LPS) injection, plasma levels of TNF-, IL-12 and IL-18 were lower in PDE3B-/- mice than WT mice. Although, as we previously reported (J. Clin. Investig. 116:3240-3251, 2006), PDE3B seems to be important in regulating certain cAMP-signaling pathways, including lipolysis, insulin- induced anti-lipolysis, and cAMP-mediated insulin secretion, PDE3B KO mice also show signs of systemic insulin resistance, most likely due to dysregulation of hepatic glucose production. They are, however, lean and not diabetic, perhaps because, in PDE3B KO mice, the presence of good BAT in EWAT depots compensates for insulin resistance, and apparently protects WAT from infiltration/accumulation of inflammatory effectors.